The proteasome is the most complex, important, and intricately regulated protease in nature. When substrates dock onto the proteasome they may undergo a variety of fates in addition to being degraded. Ubiquitin may be removed from the substrate before degradation is initiated, pre-empting degradation. Alternatively, preexisting chains on the substrate may be extended to promote degradation. Some substrate-bound chains can also shut down or slow proteasome activity, through manipulating its conformational state. Chain editing factors also regulate the processivity of the proteasome. In addition, substrate recognition can be mediated by any of six distinct proteasome-associated ubiquitin receptors. These systems were originally revealed mainly in yeast, by us and by other groups, but are conserved in eukaryotes and now understood to be relevant to significant diseases, including cancer and ALS. In this proposal we attempt to define the underlying rules of these editing processes as well as the specific sites in the proteasome that are critical for chain binding and editing. There are two distinct pathways by which a key chain-disassembling factor of the proteasome, USP14, is activated, and both are now defined genetically. One pathway provides for control via AKT-dependent signaling pathways, the other for control by the proteasome. Both pathways will be explored here. Remarkably we have found that the machinery for the activation of Ubp6 (the yeast ortholog of USP14) is identical to that for a substrate- and Ubp6-dependent inhibition of the proteasome itself that we previously reported. This linchpin thus provides for reciprocal regulation between Ubp6 and the proteasome. We will try to achieve deeper insights in these unique pathways by combining genetics, biochemistry, structural biology, and proteomics. We will also investigate USP14/Ubp6 substrate specificity, which, according to our recent work, is completely novel, as these enzymes show a dramatic preference for substrates modified by more than one ubiquitin chain. Another powerful regulator of substrate turnover is Hul5 (whose mammalian ortholog is UBE3C), a ligase that we found to be associated with the proteasome. Hul5 is important for stress resistance and is thought to act by extending proteasome-bound ubiquitin chains. We will identify favored substrates of Hul5 and UBE3C by proteomics and use these to address mechanistic models for these enzymes. We will also clarify the physiology of UBE3C and its close paralog UBE3B, which have both been linked to human disease, and attempt to explain their physiology in terms of specific ubiquitination events. Finally, to better understand how the proteasome processes ubiquitin chains we will address ubiquitin chain recognition itself, a very complex process involving to date at least six distinct receptors. Mutations in these receptors can cause ALS and have been implicated in Alzheimer?s disease. We will search for new proteasomal ubiquitin receptors whose existence is implied by our genetic data, and also investigate the relationship between the ?intrinsic ubiquitin receptors,? which are proteasome subunits, and the ?shuttle factors? that deliver ubiquitin to the proteasome.